It is well known that biochips having a flat substrate surface on which DNAs or proteins are immobilized include those prepared by Affymetrix method in which oligonucleotides are synthesized on the surface of the substrate using photolithography, and those prepared by Stanford method in which preliminarily provided probe DNAs or probe proteins are spotted so as to immobilize them on the surface of the substrate. Either type of the biochips is used such that fluorescence is detected after biological reactions with a target, and identification of the molecule or diagnosis is performed from the resulted pattern.
Among the above-mentioned two methods, the Affymetrix method has a drawback in that stable immobilization and synthesis of a long oligonucleotide are difficult because the oligonucleotide is synthesized on the surface of the substrate, and that the cost is also high. On the other hand, in the Stanford method, in order to place small spots of probe DNAs, probe proteins and the like are placed on the surface of the substrate and to immobilize the molecules to be recognized by adsorption or covalent bonds, amino groups, aldehyde groups, silanol groups or epoxy groups are covalently attached to, or polylysine is noncovalently attached to the surface of the substrate. However, it is known that since these functional groups or the polylysine are attached to the entire surface of the substrate, spots may be diffused, cross-contamination may occur due to contacts between adjacent spots, and the amounts of the immobilized molecules may differ when some spotting methods are used. It is true that uniformity in the amount and the shape (e.g., the diameter of the spots) of the spots is not attained due to the properties of the molecules per se, such as the hydrophobicity and ease of ionization thereof. In recent years, DNA chips are widespread and most of them use a glass as the material of the substrate. However, the amount of the molecules which can be bound by the modification of the silanol groups on the surface of the glass is small, and when a slide glass, a generally used substrate, is used, the amount is several nanomoles, so that the capacity of the substrate to immobilize the molecules is low. In case of immobilizing the molecules by adsorption, there is also a drawback in that non-specific adsorption strongly occurs, so that the fluorescent substances in unreacted areas, which remain even after washing after the biological reactions, decrease the S/N ratio of the detection.
Patent Literature 1: JP 2001-128683 A
Patent Literature 2: Japanese Translated PCT Patent Application Laid-open No. 2005-510440